G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111888 |
| Date | January 2007 |
| Creators | Baragli, Alessandra. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Pharmacology & Therapeutics.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002652252, proquestno: AAINR38559, Theses scanned by UMI/ProQuest. |
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